Oxidative Stress Induced Mitochondrial Protein Kinase A Mediates Cytochrome C Oxidase Dysfunction

Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.     

Iron supplementation moderates but does not cure the Belgrade anemia

Belgrade rats inherit microcytic, hypochromic anemia as an autosomalrecessive trait (gene symbol b). Erythrocytes and tissue are iron deficientin the face of elevated TIBC (total iron binding capacity) and percent ironsaturation; iron injections increased the number of erythrocytes but theirappearance remained abnormal. We have investigated iron supplements toimprove husbandry of b/b rats and to learn more about the underlying defectand its tissue distribution. Weekly IM (intramuscular) injections ofiron–dextran (Imferon at 30 mg kg) improved the anemia but did not alter thered cell morphology. Certain diets also improved the health of b/b rats whencompared to standard rat chows by the criteria of weight, survival toadulthood, hematology and reproduction. The critical nutritional factorturned out to be iron bioavailability, with ferrous iron added to the dietimproving the health of Belgrade rats without affecting the underlyingerythroid defect. Tissue iron measurements after dietary or parenteralsupplementation confirmed the iron deficient status of untreated b/b rats andestablished that dietary ferrous iron partially relieved this deficiency,with injections leading to greater amounts of tissue iron. Serum iron andTIBC were also found to be elevated in untreated b/b rats, with dietarysupplementation decreasing but not eliminating the elevation in TIBC. Thesestudies indicate that iron supplements can improve the health of b/b ratswithout altering the underlying defect and also suggest that the mutationcould alter iron uptake in the GI (gastrointestinal) tract.     

Urea production and accumulation in the green toad, Bufo viridis

The toad, Bufo viridis , can live for several months without access to free water, absorbing soil-bound water down a water-potential gradient created, mainly, by accumulating urea in its body fluids. We investigated if the retention of urine was sufficient to account for the rate of accumulation or if an increased rate of urea production was needed in order to do so. The basal rate of urea production in unfed animals in the absence of osmotic stress was estimated by two methods; first, analysis of the bathing medium and, secondly, collection and analysis of urine at two-hourly intervals. This was then repeated with animals fed a weight-maintaining diet. Generally similar results were obtained by either method in both fed and unfed animals, although higher urea production rates were found in the former. Although it had been planned to apply the short interval method to toads with free access to water, the control condition for toads transferred to soil, it proved to be impracticable. Some animals did not bathe for almost a day, during which time minute quantities of urine were obtained. Larger volumes were only produced during or after bathing. Consequently, animals which were partially immersed in water were substituted as controls. Total urea content was determined in these and in toads after a week on soil. The calculated increase was compared to that which could be expected from urine retention. It was found that urea accumulated at more than twice the predicted rate. When rates of accumulation were calculated over longer periods, urine retention alone was sufficient to account for them within three weeks on soil, the usual period required for acclimation. We concluded that B. viridis increased its rate of urea production only for a short period, until a favourable water potential gradient was achieved.     

Proteomic profiling of urine for the detection of colon cancer

Background

Colorectal cancer is the second most common cause of cancer related death in the developed world. To date, no blood or stool biomarkers with both high sensitivity and specificity for potentially curable early stage disease have been validated for clinical use. SELDI and MALDI profiling are being used increasingly to search for biomarkers in both blood and urine. Both techniques provide information predominantly on the low molecular weight proteome (<15 kDa). There have been several reports that colorectal cancer is associated with changes in the serum proteome that are detectable by SELDI and we hypothesised that proteomic changes would also be detectable in urine.

Results

We collected urine from 67 patients with colorectal cancer and 72 non-cancer control subjects, diluted to a constant protein concentration and generated MALDI and SELDI spectra. The intensities of 19 peaks differed significantly between cancer and non-cancer patients by both t-tests and after adjusting for confounders using multiple linear regressions. Logistic regression classifiers based on peak intensities identified colorectal cancer with up to 78% sensitivity at 87% specificity. We identified and independently quantified 3 of the discriminatory peaks using synthetic stable isotope peptides (an 1885 Da fragment of fibrinogen and hepcidin-20) or ELISA (β2-microglobulin).

Conclusion

Changes in the urine proteome may aid in the early detection of colorectal cancer.     

Compensatory effects of elevated CO<Subscript>2</Subscript> concentration on the inhibitory effects of high temperature and irradiance on photosynthetic gas exchange in carrots

We determined the interactive effects of irradiance, elevated CO₂ concentration (EC), and temperature in carrot (Daucus carota var. sativus). Plants of the cv. Red Core Chantenay (RCC) were grown in a controlled environmental plant growth room and exposed to 3 levels of photosynthetically active radiation (PAR) (400, 800, 1 200 μmol m⁻² s⁻¹), 3 leaf chamber temperatures (15, 20, 30 °C), and 2 external CO₂ concentrations (C _a), AC and EC (350 and 750 μmol mol⁻¹, respectively). Rates of net photosynthesis (P _N) and transpiration (E) and stomatal conductance (g _s ) were measured, along with water use efficiency (WUE) and ratio of internal and external CO₂ concentrations (C _i/C _a). P _N revealed an interactive effect between PAR and C _a. As PAR increased so did P _N under both C _a regimes. The g _s showed no interactive effects between the three parameters but had singular effects of temperature and PAR. E was strongly influenced by the combination of PAR and temperature. WUE was interactively affected by all three parameters. Maximum WUE occurred at 15 °C and 1 200 μmol m⁻² s^{− 1} PAR under EC. The C _i /C _a was influenced independently by temperature and C _a. Hence photosynthetic responses are interactively affected by changes in irradiance, external CO₂ concentration, and temperature. EC significantly compensates the inhibitory effects of high temperature and irradiance on P _N and WUE.     

A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue

Background  

Aromatase, the key enzyme in estrogen biosynthesis, is encoded by the Cyp19a1 gene. Thus far, 3 unique untranslated first exons associated with distinct promoters in the mouse Cyp19a1 gene have been described (brain, ovary, and testis-specific). It remains unknown whether aromatase is expressed in other mouse tissues via novel and tissue-specific promoters.     

Hormone measures in finger-prick blood spot samples: New field methods for reproductive endocrinology

Comparative endocrine studies have notably advanced understanding of ecological factors that contribute to variation in human reproductive function. Such research has relied on methodological advances that permit hormone determinations in samples that are easily and safely collected, stored, and transported, most recently on measurement of steroids in saliva. This report seeks to further expand the scope of endocrine research by demonstrating the value of blood spot samples collected by finger prick. As a sampling strategy, finger-prick blood spot collection offers the advantages of short collection time, low invasiveness, repeatability, absence of postcollection processing, low biohazard risk, and ease of sample storage and transport. We document good sample stability and present sensitive assay methods for a range of steroids and proteins (FSH, LH, PRL, T, E2, DHEAS, androstenedione, cortisol, SHGB) in blood spots that require sample volumes of 3–12 μl and display good reliability, specificity, precision, accuracy, and convertibility of results to plasma/serum equivalent concentrations. Laboratory evaluation was augmented by a feasibility study at a remote site in Papua New Guinea that confirmed validity and stability of blood spot collections under field conditions. Research applications of blood spot sampling are illustrated with a series of studies, including cross-sectional surveys for developmental and life span endocrinology, a longitudinal, population-based developmental epidemiologic study of puberty, and serial sampling in a dynamic study of neuroendocrine response to suckling. We conclude that the sampling features and wide range of measurable biomolecules of blood spots do constitute a methodological advance for endocrine research. Am J Phys Anthropol 104:1–21, 1997. © 1997 Wiley-Liss, Inc.     

Diagnostic accuracy of case-finding questions to identify perinatal depression

Background:

Guidelines for perinatal mental health care recommend the use of two case-finding questions about depressed feelings and loss of interest in activities, despite the absence of validation studies in this context. We examined the diagnostic accuracy of these questions and of a third question about the need for help asked of women receiving perinatal care.

Methods:

We evaluated self-reported responses to two case-finding questions against an interviewer-assessed diagnostic standard (DSM-IV criteria for major depressive disorder) among 152 women receiving antenatal care at 26–28 weeks’ gestation and postnatal care at 5–13 weeks after delivery. Among women who answered “yes” to either question, we assessed the usefulness of asking a third question about the need for help. We calculated sensitivity, specificity and likelihood ratios for the two case-finding questions and for the added question about the need for help.

Results:

Antenatally, the two case-finding questions had a sensitivity of 100% (95% confidence interval [CI] 77%–100%), a specificity of 68% (95% CI 58%–76%), a positive likelihood ratio of 3.03 (95% CI 2.28–4.02) and a negative likelihood ratio of 0.041 (95% CI 0.003–0.63) in identifying perinatal depression. Postnatal results were similar. Among the women who screened positive antenatally, the additional question about the need for help had a sensitivity of 58% (95% CI 38%–76%), a specificity of 91% (95% CI 78%–97%), a positive likelihood ratio of 6.86 (95% CI 2.16–21.7) and a negative likelihood ratio of 0.45 (95% CI 0.25–0.80), with lower sensitivity and higher specificity postnatally.

Interpretation:

Negative responses to both of the case-finding questions showed acceptable accuracy for ruling out perinatal depression. For positive responses, the use of a third question about the need for help improved specificity and the ability to rule in depression.The occurrence of depressive symptoms during the perinatal period is well-recognized. The estimated prevalence is 7.4%–20% antenatally^1,2 and up to 19.2% in the first three postnatal months.³ Antenatal depression is associated with malnutrition, substance and alcohol abuse, poor self-reported health, poor use of antenatal care services and adverse neonatal outcomes.⁴ Postnatal depression has a substantial impact on the mother and her partner, the family, mother–baby interaction and on the longer-term emotional and cognitive development of the baby.⁵Screening strategies to identify perinatal depression have been advocated, and specific questionnaires for use in the perinatal period, such as the Edinburgh Postnatal Depression Scale,⁶ were developed. However, in their current recommendations, the UK National Screening Committee⁷ and the US Committee on Obstetric Practice⁸ state that there is insufficient evidence to support the implementation of universal perinatal screening programs. The initial decision in 2001 by the National Screening Committee to not support universal perinatal screening⁹ attracted particular controversy in the United Kingdom; some service providers subsequently withdrew resources for treatment of postnatal depression, and subsequent pressure by perinatal community practitioners led to modification of the screening guidance in order to clarify the role of screening questionnaires in the assessment of perinatal depression.¹⁰In 2007, the National Institute for Health and Clinical Excellence issued clinical guidelines for perinatal mental health care in the UK, which included guidance on the use of questionnaires to identify antenatal and postnatal depression.¹¹ In this guidance, a case-finding approach to identify perinatal depression was strongly recommended; it involved the use of two case-finding questions (sometimes referred to as the Whooley questions), and an additional question about the need for help asked of women who answered “yes” to either of the initial questions (Box 1).

Box 1:

Case-finding questions recommended for the identification of perinatal depression¹⁰

• “During the past month, have you often been bothered by feeling down, depressed or hopeless?”
• “During the past month, have you often been bothered by having little interest or pleasure in doing things?”
• A third question should be considered if the woman answers “yes” to either of the initial screening questions: “Is this something you feel you need or want help with?”

Useful case-finding questions should be both sensitive and specific so they accurately identify those with and without the condition. The two case-finding questions have been validated in primary care samples^12,13 and examined in other clinical populations^14–16 and are endorsed in recommendations by US and Canadian bodies for screening depression in adults.^17,18 However, at the time the guidance from the National Institute for Health and Clinical Excellence was issued, there were no validation studies conducted in perinatal populations. A recent systematic review¹⁹ identified one study conducted in the United States that validated the two questions against established diagnostic criteria in 506 women attending well-child visits postnatally;²⁰ sensitivity and specificity of the questions were 100% and 44% respectively at four weeks. The review failed to identify studies that validated the two questions and the additional question about the need for help against a gold-standard measure.We conducted a validation study to assess the diagnostic accuracy of this brief case-finding approach against gold-standard psychiatric diagnostic criteria for depression in a population of women receiving perinatal care.     

Overstory and understory vegetation interact to alter soil community composition and activity

Aim

To test if there is an interactive effect between tree and understory species on the soil microbial community (SMC), community level physiological profiles (CLPP) and soil micro-fauna.

Method

A replicate pot experiment with five sapling tree species (Betula pendula, Betula pubescens, Sorbus aucuparia, Quercus petraea and Pinus sylvestris) and a no-tree treatment with and without Calluna vulgaris was established. After 21?months samples were taken for phospholipid fatty acid (PLFA) analysis, CLPP and soil microfauna assessment.

Results

There was an interactive effect of tree species and Calluna on the SMC, CLPP and nematode densities. Calluna addition changed the SMC composition (increase in fungal PLFAs) and the CLPP (lower utilisation of most carbon sources but greater utilisation of phenolic acids). A multivariate test for homogeneity of dispersion showed that while Calluna addition resulted in the presence of an altered microbial composition, it did not result in there being less variability among the samples with Calluna than among the samples without Calluna. Sapling trees with Calluna present grew less well than trees without Calluna. Structural equation modelling showed that it is possible that Calluna had an indirect effect on the SMC via below-ground tree biomass as well as a direct effect.

Conclusion

Interactions between trees and understory vegetation can impact on the composition of soil biota and their activity.     

Ascorbic acid enhancement of organogenesis in tobacco callus

The effect of ascorbic acid on growth and shoot formation in callus cultures of tobacco (Nicotiana tabacum L.) was investigated, using young (4–12 subcultures) and old (more than 30 subcultures) tissue. It was found that ascorbate, at levels of 4–8×10^-4M, enhanced shoot formation in both young and old callus. Treatment with ascorbate also speeded up the shoot-forming process. In addition, ascorbate completely reversed the inhibition of shoot formation by gibberellic acid in young callus, but was less effective in old callus.